Asthma is characterized by airway hyper-responsiveness, inflammation, and dysregulation of innate and adaptive immunity. Interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-13 (IL-13) are characteristic cytokines upregulated in the type 2 helper T cell (Th2) endotype which is the most common form of asthma Expression of these cytokines is driven, in part, by the zinc-finger transcriptional activator, GATA3, which is expressed in different lung cells, such as mast cells, macrophages, and epithelial cells. Indeed, targeting GATA3 is a promising therapeutic avenue to treat asthmatic patients with the Th2 endotype. Several methods to block GATA3 expression levels by knockdown have been previously investigated, including antisense, siRNA, and DNA enzyme (Dz) based approaches. Among these gene-regulation strategies, Dz-based targeting of GATA3 has shown the greatest promise having passed phase II human trials as a treatment for moderate asthma. The efficacy of Dzs is due to the fact that these molecules are short DNA oligonucleotide that catalytically degrades target mRNA, and thus are more efficient compared to antisense and avoiding the immunogenicity and stability issues of RNAi. Through a highly interdisciplinary collaboration between Dr. Salaita (co-PI) and Dr. Wongtrakool (co-PI), the team has obtained preliminary data showing that GATA3- cleaving DNAzyme nanoparticles (DzNP) are 100-fold more active at cleaving GATA-3 compared to soluble Dzs. Importantly, DzNPs also demonstrate significant efficacy in a Th2 mouse model of asthma. The goal of this proposal is to determine why DzNPs mediate improved efficacy compared to soluble Dzs by elucidating the mechanism of how GATA3-DzNPs differ from Dzs in terms of internalization, cell targeting, and stability. Our premise is that DzNPs are more effective compared to soluble Dzs due to the selective delivery of their payload in scavenger receptor expressing cells, which are upregulated in the Th2 endotype. The long-term goal of this proposal is to pave the way for the rational design of improved treatments of lung disease.